Bone morphogenetic protein (BMP)-17 and BMP-18 compositions

ABSTRACT

Purified BMP-17 and BMP-18 proteins and processes for producing them are disclosed. DNA molecules encoding the BMP-17 and BMP-18 proteins are also disclosed. The proteins may be used in the treatment of bone, cartilage, other connective tissue defects and disorders, including tendon, ligament and meniscus, in wound healing and related tissue repair, as well as for treatment of disorders and defects to tissues which include epidermis, nerve, muscle, including cardiac muscle, and other tissues and wounds, and organs such as liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidney tissue. The proteins may also be useful for the induction of growth and/or differentiation of undifferentiated embryonic and stem cells.

This application is a division of application Ser. No. 08/987,904, filedDec. 10, 1997 now U.S. Pat. No. 6,027,917.

The present invention relates to a novel family of purified proteinsdesignated as Bone Morphogenetic Proteins (BMP)-17 and BMP-18, relatedproteins, DNA encoding them, and processes for obtaining them. Theseproteins may be used to induce bone and/or cartilage or other connectivetissue formation, and in wound healing and tissue repair. These proteinsmay also be used for augmenting the activity of other bone morphogeneticproteins.

BACKGROUND OF THE INVENTION

The search for the molecule or molecules responsible for the bone-,cartilage-, and other connective tissue-inductive activity present inbone and other tissue extracts has led to the discovery of a novel setof molecules called the Bone Morphogenetic Proteins (BMPs). Thestructures of several proteins, designated BMP-1 through BMP-16 havepreviously been elucidated. The unique inductive activities of theseproteins, along with their presence in bone, suggests that they areimportant regulators of bone repair processes, and may be involved inthe normal maintenance of bone tissue. There is a need to identifywhether additional proteins, particularly human proteins, exist whichplay a role in these processes. The present invention relates to theidentification of such a novel human protein, which the inventors havedesignated human BMP-17 and BMP-18.

Human BMP-17 and BMP-18 appear to be human homologs of a murine proteincalled Lefty. The nucleotide and amino acid sequences of murine Leftyare described in Zhou et al., Nature, 361:543-547 (1993). The murineLefty gene has been described as being expressed in the mouse nodeduring gastrulation. A related human protein, designated endometrialbleeding associated factor [EBAF] was published in Kothapelli et al., J.Clin. Invest., 99:2342-2350 (1997).

SUMMARY OF THE INVENTION

As used herein, the term BMP-17 and BMP-18 proteins refer to the humanBMP-17 and BMP-18 proteins, having the amino acid sequences specified inSEQUENCE (SEQ) ID NO:2 and SEQ ID NO: 4, as well as DNA sequencesencoding the BMP-17 and BMP-18 proteins, such as the native humansequences shown in SEQ ID NO: 1 and SEQ ID NO: 3. Also included arenaturally occurring allelic sequences of SEQ ID NO:1 and 3, andequivalent degenerative codon sequences of the above.

The BMP-17 (SEQ ID NO: 1) and BMP-18 (SEQ ID NO: 3) DNA sequences andamino acid sequences (SEQ ID NO: 2 and 4, respectively) are set forth inthe Sequence Listings. BMP-17 and BMP-18 proteins may be capable ofinducing the formation of cartilage, bone, or other connective tissue,or combinations thereof. The cartilage and/or bone and/or otherconnective tissue formation activity in the rat bone formation assaydescribed below. BMP-17 and BMP-18 proteins may be further characterizedby the ability to demonstrate effects upon the growth and/ordifferentiation of embryonic cells and/or stem cells. Thus, the proteinsor compositions of the present invention may also be useful for treatingcell populations, such as embryonic cells or stem cell populations, toenhance or enrich the growth and/or differentiation of the cells.Alternatively, the proteins or compositions of the present invention mayalso be useful for maintenance of a cell population, includingdifferentiated cell populations, for example, neuronal cells, epithelialcells, dendritic cells, chondrocytes, osteocytes, muscle cells or cellsof other differentiated phenotypes.

Human BMP-17 proteins may be produced by culturing a cell transformedwith a DNA sequence comprising nucleotide a DNA sequence encoding themature BMP-17 polypeptide, comprising nucleotide #427 to nucleotide#1098 as shown in SEQ ID NO: 1, and recovering and purifying from theculture medium a protein characterized by the amino acid sequencecomprising amino acids #1 to #224 as shown in SEQ ID NO:2 substantiallyfree from other proteinaceous materials with which it is co-produced.For production in mammalian cells, the DNA sequence further comprises aDNA sequence encoding a suitable propeptide 5′ to and linked in frame tothe nucleotide sequence encoding the mature BMP-17-related polypeptide.

Human BMP-18 proteins may be produced by culturing a cell transformedwith a DNA sequence comprising nucleotide a DNA sequence encoding themature BMP-18 polypeptide, comprising nucleotide #406 to nucleotide#1098 as shown in SEQ ID NO: 3, and recovering and purifying from theculture medium a protein characterized by the amino acid sequencecomprising amino acids #1 to #231 as shown in SEQ ID NO:4 substantiallyfree from other proteinaceous materials with which it is co-produced.For production in mammalian cells, the DNA sequence further comprises aDNA sequence encoding a suitable propeptide 5′ to and linked in frame tothe nucleotide sequence encoding the mature BMP-18-related polypeptide.

The propeptide may be the native BMP-17 or BMP-18-related propeptide, ormay be a propeptide from another protein of the TGF-β superfamily. Wherethe native BMP-17 and BMP-18 propeptide is used, human BMP-17 and BMP-18may be produced by culturing a cell transformed with a DNA sequencecomprising a DNA sequence encoding the full BMP-17 and BMP-18polypeptide, comprising nucleotides #1 to #1098 as shown in SEQ ID NO:1, or nucleotides #1 to #1098 as shown in SEQ ID NO: 3, producing aprotein characterized by the amino acid sequence comprising amino acids#−142 to #224 as shown in SEQ ID NO:2, of which amino acids #−142 to −1comprise the native propeptide of human BMP-17; or #−135 to #231 of SEQID NO: 4 of which amino acids #−135 to −1 comprise the native propeptideof human BMP-18, and recovering and purifying from the culture medium aprotein characterized by the amino acid sequence comprising amino acids#1 to #224 as shown in SEQ ID NO:2, or #1 to #231 of SEQ ID NO:4,respectively, substantially free from other proteinaceous materials withwhich it is co-produced.

Based in part upon the Von Heginje signal peptide prediction algorithm,approximately the first 17 to 23 amino acids of SEQ ID NO: 2 and 4appear to be involved in signalling for the secretion of the maturepeptide. Accordingly, in one embodiment of the invention, DNA encoding asignal peptide,such as the native BMP-17 or BMP-18 signal peptide, oranother recognized signal peptide, may be linked directly to thesequence encoding the mature BMP-17 or BMP-18 peptide.

It is expected that other species, particularly human, have DNAsequences homologous to human BMP-17 and BMP-18 protein. The invention,therefore, includes methods for obtaining the DNA sequences encodinghuman BMP-17 and BMP-18 proteins, the DNA sequences obtained by thosemethods, and the human proteins encoded by those DNA sequences. Thismethod entails utilizing the human BMP-17 and BMP-18 nucleotidesequences or portions thereof to design probes to screen libraries forthe corresponding gene from other species or coding sequences orfragments thereof from using standard techniques. Thus, the presentinvention may include DNA sequences from other species, which arehomologous to human BMP-17 and BMP-18 proteins and can be obtained usingthe human BMP-17 and/or BMP-18 sequences. The present invention may alsoinclude functional fragments of the human BMP-17 and BMP-18 proteins,and DNA sequences encoding such functional fragments, as well asfunctional fragments of other related proteins. The ability of such afragment to function is determinable by assay of the protein in thebiological assays described for the assay of the BMP-17 and BMP-18proteins. DNA sequences encoding the complete mature human BMP-17 (SEQID NO: 1 and BMP-18 protein (SEQ ID NO:3) and the corresponding aminoacid sequences (SEQ ID NO:2 and 4, respectively) are set forth herein.The BMP-17 and BMP-18 proteins of the present invention, such as humanBMP-17 and BMP-18, may be produced by culturing a cell transformed withthe correlating DNA sequence, such as the human BMP-17 and BMP-18 DNAsequence, and recovering and purifying protein, such as BMP-17 orBMP-18, from the culture medium. The purified expressed protein issubstantially free from other proteinaceous materials with which it isco-produced, as well as from other contaminants. The recovered purifiedprotein is contemplated to exhibit cartilage and/or bone and/orconnective tissue formation activity. Thus, the proteins of theinvention may be further characterized by the ability to demonstratecartilage and/or bone and/or other connective tissue formation activityin the rat bone formation assay described below. BMP-17 and BMP-18proteins may be further characterized by the ability to demonstrateeffects upon the growth and/or differentiation of embryonic cells and/orstem cells. Thus, the proteins or compositions of the present inventionmay also be characterized by their ability to enhance or enrich thegrowth and/or differentiation of the cells.

Another aspect of the invention provides pharmaceutical compositionscontaining a therapeutically effective amount of human BMP-17 and/orBMP-18 protein, in a pharmaceutically acceptable vehicle or carrier.These compositions of the invention may be used in the formation ofbone. These compositions may further be utilized for the formation ofcartilage, or other connective tissue, including tendon, ligament,meniscus and other connective tissue, as well as combinations of theabove, for example regeneration of the tendon-to-bone attachmentapparatus. The compositions of the present invention, such ascompositions of human BMP-17 and/or BMP-18, may also be used for woundhealing and tissue repair. Compositions of the invention may furtherinclude at least one other therapeutically useful agent such as the BMPproteins BMP-1, BMP-2, BMP-3, BMP4, BMP-5, BMP-6 and BMP-7, disclosedfor instance in U.S. Pat. Nos.5,108,922; 5,013,649; 5,116,738;5,106,748; 5,187,076; and 5,141,905; BMP-8, disclosed in PCT publicationWO91/18098; and BMP-9, disclosed in PCT publication WO93/00432, BMP-10,disclosed in U.S. Pat. No. 5,637,480; BMP-11, disclosed in U.S. Pat. No.5,639,638, or BMP-12 or BMP-13, disclosed in U.S. Pat. No. 5,658,882,BMP-15, disclosed U.S. Pat. No. 5,635,372 and BMP-16, disclosed inco-pending patent application Ser. No. 08/715,202. Other compositionswhich may also be useful include Vgr-2, and any of the growth anddifferentiation factors [GDFs], including those described in PCTapplications WO94/15965; WO94/15949; WO95/01801; WO95/01802; WO94/21681;WO94/15966; WO95/10539; WO96/01845; WO96/02559 and others. Also usefulin the present invention may be BIP, disclosed in WO94/01557; HP00269,disclosed in JP Publication number: 7-250688; and MP52, disclosed in PCTapplication WO93/16099. The disclosures of all of the above applicationsare hereby incorporated by reference.

The compositions of the invention may comprise, in addition to a BMP-17and/or -18-related protein, other therapeutically useful agentsincluding growth factors such as epidermal growth factor (EGF),fibroblast growth factor (FGF), transforming growth factor (TGF-α andTGF-β), activins, inhibins, and insulin-like growth factor (IGF). Thecompositions may also include an appropriate matrix for instance, forsupporting the composition and providing a surface for bone and/orcartilage and/or other connective tissue growth. The matrix may provideslow release of the osteoinductive protein and/or the appropriateenvironment for presentation thereof.

The BMP-17 and/or BMP-18 containing compositions may be employed inmethods for treating a number of bone and/or cartilage and/or otherconnective tissue defects, periodontal disease and healing of varioustypes of tissues and wounds. The tissue and wounds which may be treatedinclude epidermis, nerve, muscle, including cardiac muscle, and othertissues and wounds, and other organs such as liver, lung, epithelium,brain, spleen, cardiac, pancreas and kidney tissue. These methods,according to the invention, entail administering to a patient needingsuch bone and/or cartilage and/or other connective tissue formation,wound healing or tissue repair, an effective amount of a BMP-17 and/orBMP-18 protein. The BMP-17 and/or BMP-18 containing compositions mayalso be used to treat or prevent such conditions as osteoarthritis,osteoporosis, and other abnormalities of bone, cartilage, muscle,tendon, ligament or other connective tissue, organs such as liver, lung,epithelium, brain, spleen, cardiac, pancreas and kidney tissue, andother tissues. These methods may also entail the administration of aprotein of the invention in conjunction with at least one other BMPprotein as described above. In addition, these methods may also includethe administration of a BMP-17 and/or BMP-18 protein with other growthfactors including EGF, FGF, TGF-α, TGF-β, activin, inhibin and IGF.

Still a further aspect of the invention are DNA sequences coding forexpression of a BMP-17 and/or BMP-18 protein. Such sequences include thesequence of nucleotides in a 5′ to 3′ direction illustrated in SEQ IDNO: 1 or SEQ ID NO: 3, DNA sequences which, but for the degeneracy ofthe genetic code, are identical to the DNA sequence SEQ ID NO: 1 or SEQID NO: 3, and encode the protein of SEQ ID NO: 2 or SEQ ID NO: 4.Further included in the present invention are DNA sequences whichhybridize under stringent conditions with the DNA sequence of SEQ ID NO:1 or SEQ ID NO: 3 and encode a protein having the ability to induceformation of cartilage, bone and/or other connective tissue, organs suchas liver, lung, epithelium, brain, spleen, cardiac, pancreas and kidneytissue, or other activities disclosed for BMP-17 and BMP-18. PreferredDNA sequences include those which hybridize under stringent conditions[For example, see conditions described in Maniatis et al, MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982),pages 387 to 389]. It is generally preferred that such DNA sequencesencode a polypeptide which is at least about 80% homologous, and morepreferably at least about 90% homologous, to the conserved C-terminalcysteine structure of the amino acid sequence shown in SEQ ID NO:2 orSEQ ID NO: 4. Finally, allelic or other variations of the sequences ofSEQ ID NO: 1 or SEQ ID NO: 3, whether or not such nucleotide changesresult in changes in the peptide sequence, where the peptide sequenceretains one or more BMP-17 and/or BMP-18 activity, are also included inthe present invention. The present invention also includes fragments ofthe DNA sequence of BMP-17 and/or BMP-18 shown in SEQ ID NO: 1 or SEQ IDNO: 3 which encode a polypeptide which retains the activity of BMP-17and/or BMP-18 protein.

The DNA sequences of the present invention are useful, for example, asprobes for the detection of mRNA encoding BMP-17 and/or BMP-18 in agiven cell population. Thus, the present invention includes methods ofdetecting or diagnosing genetic disorders involving the BMP-17 and/orBMP-18 gene, or disorders involving cellular, organ or tissue disordersin which BMP-17 and/or BMP-18 is irregularly transcribed or expressed.The DNA sequences may also be useful for preparing vectors for genetherapy applications as described below.

A further aspect of the invention includes vectors comprising a DNAsequence as described above in operative association with an expressioncontrol sequence therefor. These vectors may be employed in a novelprocess for producing a BMP-17 and/or BMP-18 protein of the invention inwhich a cell line transformed with a DNA sequence encoding a BMP-17and/or BMP-18 protein in operative association with an expressioncontrol sequence therefor, is cultured in a suitable culture medium anda BMP-17 and/or BMP-18-related protein is recovered and purifiedtherefrom. This process may employ a number of known cells bothprokaryotic and eukaryotic as host cells for expression of thepolypeptide. The vectors may be used in gene therapy applications. Insuch use, the vectors may be transfected into the cells of a patient exvivo, and the cells may be reintroduced into a patient. Alternatively,the vectors may be introduced into a patient in vivo through targetedtransfection.

Still a further aspect of the invention are BMP-17 and/or BMP-18proteins or polypeptides. Such polypeptides are characterized by havingan amino acid sequence including the sequence illustrated in SEQ ID NO:2 or SEQ ID NO: 4, variants of the amino acid sequence of SEQ ID NO: 2or SEQ ID NO: 4, including naturally occurring allelic variants, andother variants in which the protein retains the ability to induce theformation of cartilage and/or bone and/or other connective tissue, orother organs such as liver, lung, epithelium, brain, spleen, cardiac,pancreas and kidney tissue, or other activity characteristic of BMP-17and/or BMP-18. Preferred polypeptides include a polypeptide which is atleast about 80% homologous, and more preferably at least about 90%homologous, to the mature human BMP-17 and/or BMP-18 amino acid sequenceshown in SEQ ID NO:2 or SEQ ID NO: 4. Finally, allelic or othervariations of the sequences of SEQ ID NO: 2 or SEQ ID NO: 4, whethersuch amino acid changes are induced by mutagenesis, chemical alteration,or by alteration of DNA sequence used to produce the polypeptide, wherethe peptide sequence still has BMP-17 and/or BMP-18 activity, are alsoincluded in the present invention. The present invention also includesfragments of the amino acid sequence of BMP-17 and/or BMP-18 shown inSEQ ID NO: 2 or SEQ ID NO: 4 which retain the activity of BMP-17 and/orBMP-18 protein.

The purified proteins of the present inventions may be used to generateantibodies, either monoclonal or polyclonal, to human BMP-17 and/orBMP-18 and/or other BMP-17 and/or BMP-18-related proteins, using methodsthat are known in the art of antibody production. Thus, the presentinvention also includes antibodies to human BMP-17 and/or BMP-18 and/orother related proteins. The antibodies may be useful for purification ofBMP-17 and/or BMP-18 and/or other BMP-17 and/or BMP-18 related proteins,or for inhibiting or preventing the effects of BMP-17 and/or BMP-18related proteins. The BMP-17 and/or BMP-18 protein and related proteinsmay be useful for inducing the growth and/or differentiation ofembryonic cells and/or stem cells. Thus, the proteins or compositions ofthe present invention may also be useful for treating relativelyundifferentiated cell populations, such as embryonic cells or stem cellpopulations, to enhance or enrich the growth and/or differentiation ofthe cells. The treated cell populations may be useful for implantationand for gene therapy applications.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a nucleotide sequence containing nucleotide sequenceencoding the entire mature human BMP-17 polypeptide.

SEQ ID NO:2 is a amino acid sequence containing the mature human BMP-17polypeptide sequence.

SEQ ID NO: 3 is a nucleotide sequence containing nucleotide sequenceencoding the entire mature human BMP-18 polypeptide.

SEQ ID NO: 4 is a amino acid sequence containing the mature human BMP-17polypeptide sequence.

DETAILED DESCRIPTION OF THE INVENTION

The human BMP-17 and BMP-18 sequences of the present invention may beobtained using the whole or fragments of the murine Lefty DNA sequence,or a partial human BMP-17 or BMP-18 sequence, as a probe. Thus, thehuman BMP-17 and BMP-18 DNA sequence comprise the DNA sequence ofnucleotides #1 to #1098 of SEQ ID NO: 1 or #1 to #1098 of SEQ ID NO:3.The human BMP-17 and BMP-18 proteins comprise the sequences of aminoacids #−142 to #224 of SEQ ID NO: 2, or #−135 to #231 of SEQ ID NO: 4,respectively. The mature human BMP-17 and BMP-18 proteins are encoded bynucleotides #427 to #1098 of SEQ ID NO:1 and #406 to #1098 of SEQ IDNO:3, respectively, and comprises the sequence of amino acids #1 to #224of SEQ ID NO:2, or #1 to #231 of SEQ ID NO: 4, respectively.

It is expected that human BMP-17 and BMP-18 polypeptides, as expressedby mammalian cells such as CHO cells, exists as a heterogeneouspopulation of active species of BMP-17 and BMP-18 proteins with varyingN-termini. It is expected that active species will comprise an aminoacid sequence beginning with the residue at amino acid #109 or #124 ofSEQ ID NO:2 or #116 or #131 of SEQ ID NO: 4, respectively, or willcomprise additional amino acid sequence further in the N-terminaldirection. Thus, it is expected that DNA sequences encoding activeBMP-17 and BMP-18 polypeptides will comprise a nucleotide sequencecomprising nucleotides #1, #232, #406, #427, #751 or 190 796 to #1059 or#1098 of SEQ ID NO: 1, or #1, #232, #406, #427, #751 or #796 to #1059 or#1098 of SEQ. ID NO: 3, respectively. Accordingly, active species ofhuman BMP-17 and BMP-18 are expected to include those comprising aminoacids #−142, #−65, #−7, #1, #109 or #124 to #211 or #224 of SEQ ID NO:2,or #−135, #−58, #1, #8, #116 or #131 to #218 or #231 of Seq ID NO:4,respectively.

A host cell may be transformed with a coding sequence encoding apropeptide suitable for the secretion of proteins by the host cell islinked in proper reading frame to the coding sequence for the matureBMP-17 and BMP-18 protein. For example, see U.S. Pat. No. 5,658,882, inwhich the propeptide of BMP-2 is fused to the DNA encoding a matureBMP-12 protein. The disclosure of this reference is hereby incorporatedby reference. Thus, the present invention includes chimeric DNAmolecules comprising a DNA sequence encoding a propeptide from a memberof the TGF-β superfamily of proteins, other than BMP-17 and BMP-18, islinked in correct reading frame to a DNA sequence encoding human BMP-17or BMP-18 protein, or a related protein. The term “chimeric” is used tosignify that the propeptide originates from a different polypeptide thanthe native BMP-17 or BMP-18 protein.

The N-terminus of one active species of human BMP-17 is expected to beexperimentally determined by expression in E. coli to be as follows:[M]ARVTV. Thus, it appears that the N-terminus of this species of BMP-17is at amino acid #1 of SEQ ID NO: 1, and a DNA sequence encoding saidspecies of BMP-17 would comprise nucleotides #427 to #1098 of SEQ IDNO: 1. The apparent molecular weight of human BMP-17 monomer is expectedto be experimentally determined by SDS-PAGE to be approximately 24.8 kDon a Novex 16% tricine gel. The human BMP-17 protein is expected toexist as a clear, colorless solution in 0.1% trifluoroacetic acid.

It is expected that other BMP-17 proteins, as expressed by mammaliancells such as CHO cells, also exist as a heterogeneous population ofactive species of BMP-17-related protein with varying N-termini. Forexample, it is expected that active species of human BMP-17 protein willcomprise an amino acid sequence beginning with the cysteine residue atamino acid #109 or the glutamic acid residue at position #124 of SEQ IDNO 2 or will comprise additional amino acid sequence further in theN-terminal direction. Thus, it is expected that DNA sequences encodingactive BMP-17 proteins include those which comprise a nucleotidesequence comprising nucleotides #1, 232, 406, 427, 751 or 796 to #1059or 1098 of SEQ ID NO: 1. Accordingly, active human BMP-17 proteinsinclude those comprising amino acids #−142, −65, −7, 1, 109 or 124 to#211 or 224 of SEQ ID NO: 2.

The N-terminus of one active species of human BMP-18 is expected to beexperimentally determined by expression in E. coli to be as follows:[M]LSPRS. Thus, it appears that the N-terminus of this species of BMP-18is at amino acid #1 of SEQ ID NO: 3, and a DNA sequence encoding saidspecies of BMP-18 would comprise nucleotides #406 to #1098 of SEQ IDNO:3. The apparent molecular weight of human BMP-18 monomer is expectedto be experimentally determined by SDS-PAGE to be approximately 25.6 kDon a Novex 16% tricine gel. The human BMP-18 protein is expected toexist as a clear, colorless solution in 0.1% trifluoroacetic acid.

It is expected that other BMP-18 proteins, as expressed by mammaliancells such as CHO cells, also exist as a heterogeneous population ofactive species of BMP-18-related protein with varying N-termini. Forexample, it is expected that active species of human BMP-18 protein willcomprise an amino acid sequence beginning with the cysteine residue atamino acid #116 or the glutamic acid residue at position #131 of SEQ IDNO:4 or will comprise additional amino acid sequence further in theN-terminal direction. Thus, it is expected that DNA sequences encodingactive BMP-18 proteins include those which comprise a nucleotidesequence comprising nucleotides #1, 232, 406, 427, 751 or 796 to #1059or 1098 of SEQ ID NO:3. Accordingly, active human BMP-18 proteinsinclude those comprising amino acids #31 135, −58, 1, 8, 116 or 131 to#218 or 231 of SEQ ID NO: 4.

The BMP-17 or BMP-18 proteins of the present invention, includepolypeptides having a molecular weight of about 24.8 to 25.6 kD inmonomeric form, said polypeptide comprising the amino acid sequence ofSEQ ID NO: 2 or SEQ ID NO: 4, respectively, and having the ability toinduce the formation of cartilage and/or bone and/or other connectivetissue in the Rosen-Modified Sampath-Reddi ectopic implant assay,described in the examples.

The BMP-17 or BMP-18 proteins recovered from the culture medium arepurified by isolating them from other proteinaceous materials from whichthey are co-produced and from other contaminants present. BMP-17 orBMP-18 proteins may be characterized by the ability to induce theformation of cartilage and/or bone and/or other connective tissue andother tissue repair and differentiation, for example, in the rat boneformation assay described below. In addition, BMP-17 or BMP-18 proteinsmay be further characterized by their effects upon the growth and/ordifferentiation of embryonic cells and/or stem cells. Thus, the proteinsor compositions of the present invention may be characterized by theembryonic stem cell assay described below.

The BMP-17 or BMP-18 proteins provided herein also include factorsencoded by the sequences similar to those of SEQ ID NO: 1 or SEQ ID NO:3, but into which modifications or deletions are naturally provided(e.g. allelic variations in the nucleotide sequence which may result inamino acid changes in the polypeptide) or deliberately engineered. Forexample, synthetic polypeptides may wholly or partially duplicatecontinuous sequences of the amino acid residues of SEQ ID NO:2 or SEQ IDNO: 4. These sequences, by virtue of sharing primary, secondary, ortertiary structural and conformational characteristics with bone growthfactor polypeptides of SEQ ID NO: 2 or SEQ ID NO: 4 may possessbiological properties in common therewith. It is know, for example thatnumerous conservative amino acid substitutions are possible withoutsignificantly modifying the structure and conformation of a protein,thus maintaining the biological properties as well. For example, it isrecognized that conservative amino acid substitutions may be made amongamino acids with basic side chains, such as lysine (Lys or K), arginine(Arg or R) and histidine (His or H); amino acids with acidic sidechains, such as aspartic acid (Asp or D) and glutamic acid (Glu or E);amino acids with uncharged polar side chains, such as asparagine (Asn orN), glutamine (Gln or Q), serine (Ser or S), threonine (Thr or T), andtyrosine (Tyr or Y); and amino acids with nonpolar side chains, such asalanine (Ala or A), glycine (Gly or G), valine (Val or V), leucine (Leuor L), isoleucine (Ile or I), proline (Pro or P), phenylalanine (Phe orF), methionine (Met or M), tryptophan (Trp or W) and cysteine (Cys orC). Thus, these modifications and deletions of the native BMP-17 orBMP-18 may be employed as biologically active substitutes fornaturally-occurring BMP-17 or BMP-18 and other polypeptides intherapeutic processes. It can be readily determined whether a givenvariant of BMP-17 or BMP-18 maintains the biological activity of BMP-17or BMP-18 by subjecting both BMP-17 or BMP-18 and the variant of BMP-17or BMP-18 to the assays described in the examples.

Other specific mutations of the sequences of BMP-17 or BMP-18 proteinsdescribed herein involve modifications of glycosylation sites. Thesemodifications may involve O-linked or N-linked glycosylation sites. Forinstance, the absence of glycosylation or only partial glycosylationresults from amino acid substitution or deletion at asparagine-linkedglycosylation recognition sites. The asparagine-linked glycosylationrecognition sites comprise tripeptide sequences which are specificallyrecognized by appropriate cellular glycosylation enzymes. Thesetripeptide sequences are either asparagine-X-threonine orasparagine-X-serine, where X is usually any amino acid. A variety ofamino acid substitutions or deletions at one or both of the first orthird amino acid positions of a glycosylation recognition site (and/oramino acid deletion at the second position) results in non-glycosylationat the modified tripeptide sequence. Additionally, bacterial expressionof BMP-17 and BMP-18-related protein will also result in production of anon-glycosylated protein, even if the glycosylation sites are leftunmodified.

The present invention also encompasses the novel DNA sequences, free ofassociation with DNA sequences encoding other proteinaceous materials,and coding for expression of BMP-17 and BMP-18 proteins. These DNAsequences include those depicted in SEQ ID NO:1 or SEQ ID NO: 3 in a 5′to 3′ direction and those sequences which hybridize thereto understringent hybridization washing conditions [for example, 0.1×SSC, 0.1%SDS at 65° C.; see, T. Maniatis et al, Molecular Cloning (A LaboratoryManual), Cold Spring Harbor Laboratory (1982), pages 387 to 389] andencode a protein maintaining one or more of the activities disclosedherein for BMP-17 or BMP-18. These DNA sequences also include thosewhich comprise the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 andthose which hybridize thereto under stringent hybridization conditionsand encode a protein which maintain the other activities disclosed forBMP-17 or BMP-18.

Similarly, DNA sequences which code for BMP-17 or BMP-18 polypeptidescoded for by the sequences of SEQ ID NO: 1 or SEQ ID NO:3, respectively,or BMP-17 or BMP-18 polypeptides which comprise the amino acid sequenceof SEQ ID NO: 2 or SEQ ID NO: 4, but which differ in codon sequence dueto the degeneracies of the genetic code or allelic variations(naturally-occurring base changes in the species population which may ormay not result in an amino acid change) also encode the novel factorsdescribed herein. Variations in the DNA sequences of SEQ ID NO: 1 or SEQID NO: 3 which are caused by point mutations or by induced modifications(including insertion, deletion, and substitution) to enhance theactivity, half-life or production of the polypeptides encoded are alsoencompassed in the invention.

Another aspect of the present invention provides a novel method forproducing BMP-17 or BMP-18 polypeptides. The method of the presentinvention involves culturing a suitable cell line, which has beentransformed with a DNA sequence encoding a BMP-17 or BMP-18 polypeptideof the invention, under the control of known regulatory sequences. Thetransformed host cells are cultured and the BMP-17 or BMP-18polypeptides recovered and purified from the culture medium. Thepurified polypeptides are substantially free from other proteins withwhich they are co-produced as well as from other contaminants.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). The selection of suitable mammalian hostcells and methods for transformation, culture, amplification, screening,product production and purification are known in the art. See, e.g.,Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively,Kaufman et al, Mol. Cell. Biol., 5(7):1650 (1985) or Howley et al, U.S.Pat. No. 4,419,446. Another suitable mammalian cell line, which isdescribed in the accompanying examples, is the monkey COS-1 cell line.The mammalian cell CV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli and the like may also be employed in this method. Forexpression of the protein in bacterial cells, DNA encoding thepropeptide of BMP-17 or BMP-18 is generally not necessary.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

Another aspect of the present invention provides vectors for use in themethod of expression of these novel BMP-17 or BMP-18 polypeptides.Preferably the vectors contain the full novel DNA sequences describedabove which encode the novel factors of the invention. Additionally, thevectors contain appropriate expression control sequences permittingexpression of the BMP-17 or BMP-18 polypeptide sequences. Alternatively,vectors incorporating modified sequences as described above are alsoembodiments of the present invention. Additionally, the sequence of SEQID NO:1 or SEQ ID NO: 3 or other sequences encoding BMP-17 or BMP-18polypeptides could be manipulated to express a mature BMP-17 or BMP-18polypeptide by deleting BMP-17 or BMP-18 propeptide sequences andreplacing them with sequences encoding the complete propeptides of otherBMP proteins or members of the TGF-β superfamily. Thus, the presentinvention includes chimeric DNA molecules encoding a propeptide from amember of the TGF-β superfamily linked in correct reading frame to a DNAsequence encoding a BMP-17 or BMP-18 polypeptide.

The vectors may be employed in the method of transforming cell lines andcontain selected regulatory sequences in operative association with theDNA coding sequences of the invention which are capable of directing thereplication and expression thereof in selected host cells. Regulatorysequences for such vectors are known to those skilled in the art and maybe selected depending upon the host cells. Such selection is routine anddoes not form part of the present invention.

A protein of the present invention, which induces cartilage and/or boneand/or other connective tissue formation in circumstances where suchtissue is not normally formed, has application in the healing of bonefractures and cartilage or other connective tissue defects in humans andother animals. Such a preparation employing a BMP-17 or BMP-18polypeptide may have prophylactic use in closed as well as open fracturereduction and also in the improved fixation of artificial joints. Denovo bone formation induced by an osteogenic agent contributes to therepair of congenital, trauma induced, or oncologic resection inducedcraniofacial defects, and also is useful in cosmetic plastic surgery. ABMP-17 or BMP-18-related polypeptide may be used in the treatment ofperiodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells, and may also support the regeneration of theperiodontal ligament and attachment apparatus, which connects bone andteeth. BMP-17 or BMP-18 polypeptides of the invention may also be usefulin the treatment of osteoporosis. A variety of osteogenic,cartilage-inducing and bone inducing factors have been described. See,e.g., European patent applications 148,155 and 169,016 for discussionsthereof.

The proteins of the invention may also be used in wound healing andrelated tissue repair. The types of wounds include, but are not limitedto burns, incisions and ulcers. (See, e.g. PCT Publication WO84/01106for discussion of wound healing and related tissue repair). It isfurther contemplated that proteins of the invention may increaseneuronal, astrocytic and glial cell survival and therefore be useful intransplantation and treatment of conditions exhibiting a decrease inneuronal survival and repair. The proteins of the invention may furtherbe useful for the treatment of conditions related to other types oftissue, such as nerve, epidermis, muscle, and other organs such asliver, lung, epithelium, brain, spleen, cardiac, pancreas and kidneytissue. The proteins of the present invention may further be useful forthe treatment of relatively undifferentiated cell populations, such asembryonic cells, or stem cells, to enhance growth and/or differentiationof the cells. The proteins of the present invention may also have valueas a dietary supplement, or as a component of cell culture media. Forthis use, the proteins may be used in intact form, or may be predigestedto provide a more readily absorbed supplement.

The proteins of the invention may also have other useful propertiescharacteristic of the TGF-β superfamily of proteins. Such propertiesinclude angiogenic, chemotactic and/or chemoattractant properties, andeffects on cells including induction of collagen synthesis, fibrosis,differentiation responses, cell proliferative responses and responsesinvolving cell adhesion, migration and extracellular matrices. Theseproperties make the proteins of the invention potential agents for woundhealing, reduction of fibrosis and reduction of scar tissue formation.

When dimerized as a homodimer or as a heterodimer with other BMPs, withother members of the TGF-β superfamily of proteins, or with inhibin-αproteins or inhibin-β proteins, the BMP-17 or BMP-18 heterodimer isexpected to demonstrate effects on the production of folliclestimulating hormone (FSH), as described further herein. It is recognizedthat FSH stimulates the development of ova in mammalian ovaries (Ross etal., in Textbook of Endocrinology, ed. Williams, p. 355 (1981) and thatexcessive stimulation of the ovaries with FSH will lead to multipleovulations. FSH is also important in testicular function. Thus, BMP-17or BMP-18 may be useful as a contraceptive based on the ability ofinhibins to decrease fertility in female mammals and decreasespermatogenesis in male mammals. Administration of sufficient amounts ofother inhibins can induce infertility in mammals. BMP-17 or BMP-18 mayalso be useful as a fertility inducing therapeutic, based upon theability of activin molecules in stimulating FSH release from cells ofthe anterior pituitary. See, for example, U.S. Pat. No. 4,798,885.BMP-17 or BMP-18 may also be useful for advancement of the onset offertility in sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs. It is further contemplated that BMP-17 or BMP-18 may be useful inmodulating hematopoiesis by inducing the differentiation of erythroidcells [see, e.g., Broxmeyer et al, Proc. Natl. Acad. Sci. USA,85:9052-9056 (1988) or Eto et al, Biochem. Biophys. Res. Comm.,142:1095-1103 (1987)], for suppressing the development of gonadal tumors[see, e.g., Matzuk et al., Nature, 360:313-319 (1992)] or for augmentingthe activity of bone morphogenetic proteins [see, e.g., Ogawa et al., J.Biol. Chem., 267:14233-14237 (1992)].

BMP-17 and BMP-18 proteins may be further characterized by their abilityto modulate the release of follicle stimulating hormone (FSH) inestablished in vitro bioassays using rat anterior pituitary cells asdescribed [see, e.g., Vale et al, Endocrinology, 91:562-572 (1972); Linget al., Nature, 321:779-782 (1986) or Vale et al., Nature, 321:776-779(1986)]. It is contemplated that the BMP-17 or BMP-18 protein of theinvention, when composed as a heterodimer with inhibin α or inhibin βchains, will exhibit regulatory effects, either stimulatory orinhibitory, on the release of follicle stimulating hormone (FSH), fromanterior pituitary cells as described [Ling et al., Nature, 321:779-782(1986) or Vale et al., Nature, 321:776-779 (1986); Vale et al,Endocrinology, 91:562-572 (1972). Therefore, depending on the particularcomposition, it is expected that the BMP-17 or BMP-18 protein of theinvention may have contrasting and opposite effects on the release offollicle stimulating hormone (FSH) from the anterior pituitary.

Activin A (the homodimeric composition of inhibin β_(A)) has been shownto have erythropoietic-stimulating activity [see e.g. Eto et al.,Biochem. Biophys. Res. Commun., 142:1095-1103 (1987) and Murata et al.,Proc. Natl. Acad. Sci. U.S.A., 85:2434-2438 (1988) and Yu et al.,Nature, 330:765-767 (1987)]. It is contemplated that the BMP-17 and -18proteins of the invention may have a similar erythropoietic-stimulatingactivity. This activity of the BMP-17 and BMP-18 proteins may be furthercharacterized by the ability of the BMP-17 and BMP-18 proteins todemonstrate erythropoietin activity in the biological assay performedusing the human K-562 cell line as described by [Lozzio et al., Blood,45:321-334 (1975) and U.S. Pat. No. 5,071,834].

A further aspect of the invention is a therapeutic method andcomposition for repairing fractures and other conditions related tocartilage and/or bone and/or other connective tissue defects orperiodontal diseases. The invention further comprises therapeuticmethods and compositions for wound healing and tissue repair. Suchcompositions comprise a therapeutically effective amount of at least oneof the BMP-17 or BMP-18-related proteins of the invention in admixturewith a pharmaceutically acceptable vehicle, carrier or matrix. It isfurther contemplated that compositions of the invention may increaseneuronal survival and therefore be useful in transplantation andtreatment of conditions exhibiting a decrease in neuronal survival.Compositions of the invention may further include at least one othertherapeutically useful agent, such as members of the TGF-β superfamilyof proteins, which includes the BMP proteins BMP-1, BMP-2, BMP-3, BMP-4,BMP-5, BMP-6 and BMP-7, disclosed for instance in U.S. Pat. No.5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; and 5,141,905;BMP-8, disclosed in PCT publication WO91/18098; BMP-9, disclosed in U.S.Pat. No. 5,661,007; BMP-10, disclosed in U.S. Pat. No. 5,637,480;BMP-11, disclosed in U.S. Pat. No. 5,639,638; BMP-12 or BMP-13,disclosed in U.S. Pat. No. 5,658,882; or BMP-15, disclosed in U.S. Pat.No. 5,635,372; or BMP-16, disclosed in co-pending patent applicationSer. No. 08/715,202. Other compositions which may also be useful includeVgr-2, and any of the growth and differentiation factors [GDFs],including those described in PCT applications WO94/15965; WO94/15949;WO95/01801; WO95/01802; WO94/21681; WO94/15966; WO95/10539; WO96/01845;WO96/02559 and others. Also useful in the present invention may be BIP,disclosed in WO94/01557; HP00269, disclosed in JP Publication number:7-250688; and MP52, disclosed in PCT application WO93/16099. Thedisclosures of the above applications are hereby incorporated byreference herein.

It is expected that human BMP-17 and BMP-18 protein may exist in natureas monomers, or as homodimers or heterodimers. To promote the formationof dimers of BMP-17 or BMP-18 and useful proteins with increasedstability, one can genetically engineer the DNA sequence of SEQ ID NO: 1or SEQ ID NO: 3 to provide one or more additional cysteine residues toincrease potential dimer formation. The resulting DNA sequence would becapable of producing a “cysteine added variant” of BMP-17 or BMP-18. Ina preferred embodiment, one would engineer the DNA sequence of SEQ IDNO: 1 or SEQ ID NO: 3 so that one or more codons may be altered to anucleotide triplet encoding a cysteine residue, such as TGT or TGC.Alternatively, one can produce “cysteine added variants” of BMP-17 orBMP-18 protein by altering the sequence of the protein at the amino acidlevel by altering one or more amino acid residues of SEQ ID NO:2 or SEQID NO: 4 to cysteine. Production of “cysteine added variants” ofproteins is described in U.S. Pat. No. 5,166,322, the disclosure ofwhich is hereby incorporated by reference. In preferred embodiments, theglutamic acid residue at position #173 of SEQ. ID NO: 2 or position #180of SEQ. ID NO: 4 is replaced by a cysteine residue.

It is expected that the proteins of the invention may act in concertwith or perhaps synergistically with other related proteins and growthfactors. Further therapeutic methods and compositions of the inventiontherefore comprise a therapeutic amount of at least one BMP-17 or BMP-18protein of the invention with a therapeutic amount of at least one othermember of the TGF-β superfamily of proteins, such as the BMP proteinsdisclosed in the applications described above. Such combinations maycomprise separate molecules of the BMP proteins or heteromoleculescomprised of different BMP moieties. For example, a method andcomposition of the invention may comprise a disulfide linked dimercomprising a BMP-17 or BMP-18 protein subunit and a subunit from one ofthe “BMP” proteins described above. Thus, the present invention includesa purified BMP-17 or BMP-18-related polypeptide which is a heterodimerwherein one subunit comprises the amino acid sequence from amino acid #1to amino acid #224 of SEQ ID NO:2 or #1 to #231 of SEQ ID NO:4, and onesubunit comprises an amino acid sequence for a bone morphogeneticprotein selected from the group consisting of BMP-2, BMP-3, BMP4, BMP-5,BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, orBMP-16. A further embodiment may comprise a heterodimer of BMP-17 orBMP-18-related moieties, for example a heterodimer of human BMP-17 andhuman BMP-18 or of human BMP-17 and the murine Letfy protein, which is ahomologue of human BMP-17 and BMP-18. Further, BMP-17 or BMP-18 proteinmaybe combined with other agents beneficial to the treatment of the boneand/or cartilage and/or other connective tissue defect, wound, or tissuein question. These agents include various growth factors such asepidermal growth factor (EGF), fibroblast growth factor (FGF), plateletderived growth factor (PDGF), transforming growth factors (TGF-α andTGF-β ), activins, inhibins, and k-fibroblast growth factor (kFGF),parathyroid hormone (PTH), parathyroid hormone related peptide (PTHrP),leukemia inhibitory factor (LIB/HILA/DA), insulin-like growth factors(IGF-I and IGF-II). Portions of these agents may also be used incompositions of the present invention. The preparation and formulationof such physiologically acceptable protein compositions, having dueregard to pH, isotonicity, stability and the like, is within the skillof the art. The therapeutic compositions are also presently valuable forveterinary applications due to the lack of species specificity in BMPproteins. Particularly domestic animals and thoroughbred horses inaddition to humans are desired patients for such treatment with theBMP-17 or BMP-18 proteins of the present invention.

The therapeutic method includes administering the composition topically,systemically, or locally as an implant or device. When administered, thetherapeutic composition for use in this invention is, of course, in apyrogen-free, physiologically acceptable form. Further, the compositionmay desirably be encapsulated or injected in a viscous form for deliveryto the site of bone, cartilage or other connective tissue or othertissue damage. Topical administration may be suitable for wound healingand tissue repair. Therapeutically useful agents other than the BMP-17or BMP-18 proteins which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the BMP composition inthe methods of the invention.

Preferably for bone and/or cartilage and/or other connective tissueformation, the composition includes a matrix capable of deliveringBMP-17 or BMP-18-related or other BMP proteins to the site of boneand/or cartilage and/or other connective tissue damage, providing astructure for the developing bone and cartilage and other connectivetissue and optimally capable of being resorbed into the body. The matrixmay provide slow release of BMP-17 or BMP-18 protein and/or other boneinductive protein, as well as proper presentation and appropriateenvironment for cellular infiltration. Such matrices may be formed ofmaterials presently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the BMP-17 or BMP-18compositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalcium phosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined, such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalcium phosphate. The bioceramics may be altered incomposition, such as in calcium-aluminate-phosphate and processing toalter pore size, particle size, particle shape, and biodegradability.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the BMP-17 orBMP-18 protein, e.g. amount of bone or other tissue weight desired to beformed, the site of bone or tissue damage, the condition of the damagedbone tissue, the size of a wound, type of damaged tissue, the patient'sage, sex, and diet, the severity of any infection, time ofadministration and other clinical factors. The dosage may vary with thetype of matrix used in the reconstitution and the types of BMP proteinsin the composition. Generally, systemic or injectable administrationwill be initiated at a dose which is minimally effective, and the dosewill be increased over a preselected time course until a positive effectis observed. Subsequently, incremental increases in dosage will be madelimiting such incremental increases to such levels that produce acorresponding increase in effect, while taking into account any adverseaffects that may appear. The addition of other known growth factors,such as IGF I (insulin like growth factor I), to the final composition,may also effect the dosage.

Progress can be monitored by periodic assessment of bone or tissuegrowth and/or repair. The progress can be monitored, for example,x-rays, histomorphometric determinations and tetracycline labeling.

The following examples illustrate practice of the present invention withrespect to human BMP-17 and human BMP-18 and other BMP-17 andBMP-18-related proteins. The skilled artisan will recognize thatnumerous variations and modifications are possible. These variations andmodifications constitute part of the present invention.

EXAMPLES Example 1 Isolation of DNA

DNA sequences encoding human BMP-17 and BMP-18 and human BMP-17 andBMP-18-related proteins may be isolated by various techniques known tothose skilled in the art using the sequence information provided in theSequence Listings.

Based on the knowledge of BMP proteins and other proteins within theTGF-β family, it is predicted that the carboxyl-terminal portion ofthese molecules (mature peptide) would exhibit greater sequenceconservation than the more amino-terminal portions (propeptide region).This sequence relationship between BMP proteins and other proteinswithin the TGF-β family enables those skilled in the art to design DNAprobes from the carboxyl-terminal encoding portion (mature peptideencoding region) of these molecules which can be utilized to identifyrelated BMP proteins and other proteins within the TGF-β family. Themature peptide encoding region of the murine lefty gene may be utilizedto identify human BMP-17 and BMP-18 and related proteins.

The bacteriophage AF02, which contains the DNA sequence encoding humanBMP-17 has been deposited with the American Type Culture Collection,10801 University Boulevard Manassas, Va. 20110-2209 ATCC under theaccession #202060 on Nov. 24, 1997. This deposit meets the requirementsof the Budapest Treaty of the International Recognition of the Depositof Microorganisms for the Purpose of Patent Procedure and Regulationsthereunder.

The bacteriophage AF04, which contains the DNA sequence encoding humanBMP-18 has been deposited with the American Type Culture Collection,10801 University Boulevard Manassas, Va. 20110-2209 ATCC under theaccession #20259 on Nov. 24, 1997. This deposit meets the requirementsof the Budapest Treaty of the International Recognition of the Depositof Microorganisms for the Purpose of Patent Procedure and Regulationsthereunder.

Based on the knowledge of other BMP proteins and other proteins withinthe TGF-β family, it is predicted that the human BMP-17 and BMP-18precursor polypeptides would be cleaved at the multibasic sequence inagreement with a proposed consensus proteolytic processing sequence ofArg-X-X-Arg corresponding to amino acids -4 to -1 of SEQ ID NO:1.Cleavage of the human BMP-17 precursor polypeptide is expected togenerate a 224 amino acid mature peptide beginning with the amino acidalanine at position #1 of SEQ ID NO:2, or a 101 amino acid maturepeptide beginning with the amino acid glutamic acid at position #124 ofSEQ ID NO: 2. Cleavage of the human BMP-18 precursor polypeptide isexpected to generate a 231 amino acid mature peptide beginning with theamino acid leucine at position #1 of SEQ ID NO:4, or a 101 amino acidmature peptide beginning with the amino acid glutamic acid at position#131 of SEQ ID NO: 4. The processing of human BMP-17 or BMP-18 into themature forms is expected to potentially involve dimerization and removalof the N-terminal region in a manner analogous to the processing of therelated protein TGF-β[Gentry et al., Molec & Cell. Biol., 8:4162 (1988);

Derynck et al. Nature, 316:701 (1985)].

It is contemplated therefore that the mature active species of humanBMP-17 and BMP-18 may comprise homodimers of two polypeptide subunits,each subunit comprising amino acids #1 to #224 of SEQ ID NO:2 or #1 to#231 of SEQ ID NO:4, respectively, with a predicted molecular weight ofapproximately 49 to 52 kD. Further active species are contemplatedcomprising at least amino acids #109 to #211 of SEQ ID NO:2 or #116 to#218 of SEQ ID NO: 4, thereby including the first and last conservedcysteine residues. As with other members of the TGF-β/BMP family ofproteins, the carboxyl-terminal portion of the human BMP-17 and BMP-18polypeptides exhibit greater sequence conservation than the moreamino-terminal portion.

The human BMP-17 (SEQ ID NO:1) or BMP-18 DNA sequence (SEQ ID NO:3), ora portion thereof, can be used as a probe to identify a human cell lineor tissue which synthesizes human BMP-17 or BMP-18 or a human BMP-17 orBMP-18-related mRNA. Briefly described, RNA is extracted from a selectedcell or tissue source and either electrophoresed on a formaldehydeagarose gel and transferred to nitrocellulose, or reacted withformaldehyde and spotted on nitrocellulose directly. The nitrocelluloseis then hybridized to a probe derived from the coding sequence of humanBMP-17 and BMP-18.

Alternatively, the human BMP-17 or BMP-18 sequence may be used to designoligonucleotide primers which will specifically amplify a portion of thehuman BMP-17 or BMP-18 or related encoding sequences. It is contemplatedthat these human BMP-17 and human BMP-18 derived primers would allow oneto specifically amplify corresponding human BMP-17 or BMP-18 or relatedencoding sequences from mRNA, cDNA or genomic DNA templates. Once apositive source has been identified by one of the above describedmethods, mRNA is selected by oligo (dT) cellulose chromatography andcDNA is synthesized and cloned in λgt10 or other λ bacteriophage vectorsknown to those skilled in the art, for example, λZAP by establishedtechniques (Toole et al., supra). It is also possible to perform theoligonucleotide primer directed amplification reaction, described above,directly on a preestablished human cDNA or genomic library which hasbeen cloned into a λ bacteriophage vector. In such cases, a librarywhich yields a specifically amplified DNA product encoding a portion ofthe human BMP-17 or BMP-18 or related protein could be screeneddirectly, utilizing the fragment of amplified human BMP-17, BMP-18 orrelated encoding DNA as a probe.

Additional methods known to those skilled in the art may be used toisolate other full-length cDNAs encoding human BMP-17 or BMP-18 orrelated proteins, or full length cDNA clones encoding BMP-17 or BMP-18or related proteins of the invention from species other than humans,particularly other mammalian species.

Example 2 W-20 Bioassays

A. Description of W-20 Cells

Use of the W-20 bone marrow stromal cells as an indicator cell line isbased upon the conversion of these cells to osteoblast-like cells aftertreatment with a BMP protein [Thies et al, Journal of Bone and MineralResearch, 5:305 (1990); and Thies et al, Endocrinology, 130:1318(1992)]. Specifically, W-20 cells are a clonal bone marrow stromal cellline derived from adult mice by researchers in the laboratory of Dr. D.Nathan, Children's Hospital, Boston, Mass. Treatment of W-20 cells withcertain BMP proteins results in (1) increased alkaline phosphataseproduction, (2) induction of PTH stimulated cAMp, and (3) induction ofosteocalcin synthesis by the cells. While (1) and (2) representcharacteristics associated with the osteoblast phenotype, the ability tosynthesize osteocalcin is a phenotypic property only displayed by matureosteoblasts. Furthermore, to date we have observed conversion of W-20stromal cells to osteoblast-like cells only upon treatment with BMPs. Inthis manner, the in vitro activities displayed by BMP treated W-20 cellscorrelate with the in vivo bone forming activity known for BMPs.

Below two in vitro assays useful in comparison of BMP activities ofnovel osteoinductive molecules are described.

B. W-20 Alkaline Phosphatase Assay Protocol

W-20 cells are plated into 96 well tissue culture plates at a density of10,000 cells per well in 200 μl of media (DME with 10% heat inactivatedfetal calf serum, 2 mM glutamine and 100 Units/ml penicillin +100 μlstreptomycin. The cells are allowed to attach overnight in a 95% air, 5%CO₂ incubator at 37° C. The 200 μl of media is removed from each wellwith a multichannel pipettor and replaced with an equal volume of testsample delivered in DME with 10% heat inactivated fetal calf serum, 2 mMglutamine and 1% penicillin-streptomycin. Test substances are assayed intriplicate. The test samples and standards are allowed a 24 hourincubation period with the W-20 indicator cells. After the 24 hours,plates are removed from the 37° C. incubator and the test media areremoved from the cells. The W-20 cell layers are washed 3 times with 200μl per well of calcium/magnesium free phosphate buffered saline andthese washes are discarded. 50 μl of glass distilled water is added toeach well and the assay plates are then placed on a dry ice/ethanol bathfor quick freezing. Once frozen, the assay plates are removed from thedry ice/ethanol bath and thawed at 37° C. This step is repeated 2 moretimes for a total of 3 freeze-thaw procedures. Once complete, themembrane bound alkaline phosphatase is available for measurement. 50 μlof assay mix (50 mM glycine, 0.05% Triton X-100, 4 mM MgCl₂, 5 mMp-nitrophenol phosphate, pH=10.3) is added to each assay well and theassay plates are then incubated for 30 minutes at 37° C. in a shakingwaterbath at 60 oscillations per minute. At the end of the 30 minuteincubation, the reaction is stopped by adding 100 μl of 0.2 N NaOH toeach well and placing the assay plates on ice. The spectrophotometricabsorbance for each well is read at a wavelength of 405 nanometers.These values are then compared to known standards to give an estimate ofthe alkaline phosphatase activity in each sample. For example, usingknown amounts of p-nitrophenol phosphate, absorbance values aregenerated. This is shown in Table I.

TABLE I Absorbance Values for Known Standards of P-Nitrophenol PhosphateP-nitrophenol phosphate umoles Mean absorbance (405 nm) 0.000 0 0.0060.261 +/− .024 0.012 0.521 +/− .031 0.018 0.797 +/− .063 0.024 1.074 +/−.061 0.030 1.305 +/− .083

Absorbance values for known amounts of BMPs can be determined andconverted to μ moles of p-nitrophenol phosphate cleaved per unit time asshown in Table II.

TABLE II Alkaline Phosphatase Values for W-20 Cells Treating with BMP-2BMP-2 concentration Absorbance Reading umoles substrate ng/ml 405nmeters per hour 0 0.645 0.024 1.56 0.696 0.026 3.12 0.765 0.029 6.250.923 0.036 12.50 1.121 0.044 25.0 1.457 0.058 50.0 1.662 0.067 100.01.977 0.080

These values are then used to compare the activities of known amounts ofBMP-17 and BMP-18 to BMP-2.

C. Osteocalcin RIA Protocol

W-20 cells are plated at 10⁶ cells per well in 24 well multiwell tissueculture dishes in 2 mls of DME containing 10% heat inactivated fetalcalf serum, 2 mM glutamine. The cells are allowed to attach overnight inan atmosphere of 95% air 5% CO₂ at 37° C. The next day the medium ischanged to DME containing 10% fetal calf serum, 2 mM glutamine and thetest substance in a total volume of 2 ml. Each test substance isadministered to triplicate wells. The test substances are incubated withthe W-20 cells for a total of 96 hours with replacement at 48 hours bythe same test medias. At the end of 96 hours, 50 μl of the test media isremoved from each well and assayed for osteocalcin production using aradioimmunoassay for mouse osteocalcin. The details of the assay aredescribed in the kit manufactured by Biomedical Technologies Inc., 378Page Street, Stoughton, Mass. 02072. Reagents for the assay are found asproduct numbers BT-431 (mouse osteocalcin standard), BT-432 (Goatanti-mouse Osteocalcin), BT-431R (iodinated mouse osteocalcin), BT-415(normal goat serum) and BT-414 (donkey anti goat IgG). The RIA forosteocalcin synthesized by W-20 cells in response to BMP treatment iscarried out as described in the protocol provided by the manufacturer.

The values obtained for the test samples are compared to values forknown standards of mouse osteocalcin and to the amount of osteocalcinproduced by W-20 cells in response to challenge with known amounts ofBMP-2. The values for BMP-2 induced osteocalcin synthesis by W-20 cellsis shown in Table III.

TABLE III Osteocalcin Synthesis by W-20 Cells BMP-2 Concentration ng/mlOsteocalcin Synthesis ng/well 0 0.8 2 0.9 4 0.8 8 2.2 17 2.7 31 3.2 625.1 125 6.5 250 8.2 500 9.4 1000 10.0

Example 3 Rosen Modified Sampath-Reddi Assay

A modified version of the rat bone formation assay described in Sampathand Reddi, Proc. Natl. Acad. Sci. USA, 80:6591-6595 (1983) is used toevaluate bone and/or cartilage and/or other connective tissue activityof BMP proteins. This modified assay is herein called the Rosen modifiedSampath-Reddi assay. The ethanol precipitation step of the Sampath-Reddiprocedure is replaced by dialyzing (if the composition is a solution) ordiafiltering (if the composition is a suspension) the fraction to beassayed against water. The solution or suspension is then equilbrated to0.1% TFA. The resulting solution is added to 20 mg of rat matrix. A mockrat matrix sample not treated with the protein serves as a control. Thismaterial is frozen and lyophilized and the resulting powder enclosed in#5 gelatin capsules. The capsules are implanted subcutaneously in theabdominal thoracic area of 21-49 day old male Long Evans rats. Theimplants are removed after 7-14 days. Half of each implant is used foralkaline phosphatase analysis [see, Reddi et al, Proc. Natl. Acad. Sci.,69:1601 (1972)].

The other half of each implant is fixed and processed for histologicalanalysis. 1 μm glycolmethacrylate sections are stained with Von Kossaand acid fuschin to score the amount of induced bone and cartlilage andother connective tissue formation present in each implant. The terms +1through +5 represent the area of each histological section of an implantoccupied by new bone and/or cartilage cells and matrix. A score of +5indicates that greater than 50% of the implant is new bone and/orcartilage produced as a direct result of protein in the implant. A scoreof +4, +3, +2, and +1 would indicate that greater than 40%, 30%, 20% and10% respectively of the implant contains new cartilage and/or bone.

Alternatively, the implants are inspected for the appearance of tissueresembling embryonic tendon, which is easily recognized by the presenceof dense bundles of fibroblasts oriented in the same plane and packedtightly together. [Tendon/ligament-like tissue is described, forexample, in Ham and Cormack, Histology (JB Lippincott Co. (1979), pp.367-369, the disclosure of which is hereby incorporated by reference].These findings may be reproduced in additional assays in whichtendon/ligament-like tissues are observed in the BMP-17 andBMP-18-related protein containing implants. The BMP-17 andBMP-18-related proteins of this invention may be assessed for activityon this assay.

Example 4 Expression of BMP-17 and BMP-18

In order to produce murine, human or other mammalian BMP-17 and BMP-18proteins, the DNA encoding it is transferred into an appropriateexpression vector and introduced into mammalian cells or other preferredeukaryotic or prokaryotic hosts by conventional genetic engineeringtechniques. The preferred expression system for biologically activerecombinant human BMP-17 and BMP-18 is contemplated to be stablytransformed mammalian cells.

One skilled in the art can construct mammalian expression vectors byemploying the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or other DNAsequences encoding BMP-17 and BMP-18 or related proteins or othermodified sequences and known vectors, such as pCD [Okayama et al., Mol.Cell Biol., 2:161 (1982)], pJL3, pJL4 [Gough et al., EMBO J., 4:645-653(1985)] and pMT2 CXM. The mammalian expression vector pMT2 CXM is aderivative of p91023(b) (Wong et al., Science 228:810-815, 1985)differing from the latter in that it contains the ampicillin resistancegene in place of the tetracycline resistance gene and further contains aXhoI site for insertion of cDNA clones. The functional elements of pMT2CXM have been described (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci.USA 82:689-693) and include the adenovirus VA genes, the SV40 origin ofreplication including the 72 bp enhancer, the adenovirus major latepromoter including a 5′ splice site and the majority of the adenovirustripartite leader sequence present on adenovirus late mRNAs, a 3′ spliceacceptor site, a DHFR insert, the SV40 early polyadenylation site(SV40), and pBR322 sequences needed for propagation in E. coli.

Plasmid pMT2 CXM is obtained by EcoRI digestion of pMT2-VWF, which hasbeen deposited with the American Type Culture Collection (ATCC),Manassas, Va. (USA) under accession number ATCC 67122. EcoRI digestionexcises the cDNA insert present in pMT2-VWF, yielding pMT2 in linearform which can be ligated and used to transform E. coli HB 101 or DH-5to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2 CXM is then constructed using loopout/inmutagenesis [Morinaga, et al., Biotechnology 84: 636 (1984). Thisremoves bases 1075 to 1145 relative to the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. In addition itinserts the following sequence:

5′PO-CATGGGCAGCTCGAG-3′SEQ ID NO:15

at nucleotide 1145. This sequence contains the recognition site for therestriction endonuclease Xho I. A derivative of pMT2CXM, termed pMT23,contains recognition sites for the restriction endonucleases PstI, EcoRI, SalI and XhoI. Plasmid pMT2 CXM and pMT23 DNA may be prepared byconventional methods.

pEMC2β1 derived from pMT21 may also be suitable in practice of theinvention. pMT21 is derived from pMT2 which is derived from pMT2-VWF. Asdescribed above EcoRI digestion excises the cDNA insert present inpMT-VWF, yielding pMT2 in linear form which can be ligated and used totransform E. Coli HB 101 or DH-5 to ampicillin resistance. Plasmid pMT2DNA can be prepared by conventional methods.

pMT21 is derived from pMT2 through the following two modifications.First, 76 bp of the 5′ untranslated region of the DHFR cDNA including astretch of 19 G residues from G/C tailing for cDNA cloning is deleted.In this process, a XhoI site is inserted to obtain the followingsequence immediately upstream from DHFR:

(SEQ ID NO:6) 5′-CTGCAGGCGAGCCTGAATTCCTCGAGCCATCATG-3′   PstI          Eco RI XhoI

Second, a unique ClaI site is introduced by digestion with EcoRV andXbaI, treatment with Klenow fragment of DNA polymerase I, and ligationto a ClaI linker (CATCGATG). This deletes a 250 bp segment from theadenovirus associated RNA (VAI) region but does not interfere with VAIRNA gene expression or function. pMT21 is digested with EcoRI and XhoI,and used to derive the vector pEMC2B1.

A portion of the EMCV leader is obtained from pMT2-ECAT1 [S. K. Jung, etal, J. Virol 63:1651-1660 (1989)] by digestion with Eco RI and pstI,resulting in a 2752 bp fragment. This fragment is digested with TaqIyielding an Eco RI-TaqI fragment of 508 bp which is purified byelectrophoresis on low melting agarose gel. A 68 bp adapter and itscomplementary strand are synthesized with a 5′ TaqI protruding end and a3′ XhoI protruding end which has the following sequence:

5′-CGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTT (SEQ ID NO:7)   TaqI GAAAAACACGATTGC-3′          XhoI

This sequence matches the EMC virus leader sequence from nucleotide 763to 827. It also changes the ATG at position 10 within the EMC virusleader to an ATT and is followed by a XhoI site. A three way ligation ofthe pMT21 Eco RI-16hoI fragment, the EMC virus EcoRI-TaqI fragment, andthe 68 bp oligonucleotide adapter TaqI-16hoI adapter resulting in thevector pEMC2p1.

This vector contains the SV40 origin of replication and enhancer, theadenovirus major late promoter, a cDNA copy of the majority of theadenovirus tripartite leader sequence, a small hybrid interveningsequence, an SV40 polyadenylation signal and the adenovirus VA I gene,DHFR and β-lactamase markers and an EMC sequence, in appropriaterelationships to direct the high level expression of the desired cDNA inmammalian cells.

The construction of vectors may involve modification of the BMP-17 andBMP-18-related DNA sequences. For instance, BMP-17 and BMP-18 cDNA canbe modified by removing the non-coding nucleotides on the 5′ and 3′ endsof the coding region. The deleted non-coding nucleotides may or may notbe replaced by other sequences known to be beneficial for expression.These vectors are transformed into appropriate host cells for expressionof BMP-17 and BMP-18-related proteins. Additionally, the sequence of SEQID NO:1, SEQ ID NO: 3 or other sequences encoding BMP-17 andBMP-18-related proteins can be manipulated to express a mature BMP-17 orBMP-18 or related protein by deleting BMP-17 or BMP-18 encodingpropeptide sequences and replacing them with sequences encoding thecomplete propeptides of other BMP proteins.

One skilled in the art can manipulate the sequences of SEQ ID NO: 1 orSEQ ID NO: 3 by eliminating or replacing the mammalian regulatorysequences flanking the coding sequence with bacterial sequences tocreate bacterial vectors for intracellular or extracellular expressionby bacterial cells. For example, the coding sequences could be furthermanipulated (e.g. ligated to other known linkers or modified by deletingnon-coding sequences therefrom or altering nucleotides therein by otherknown techniques). The modified BMP-17 or BMP-18-related coding sequencecould then be inserted into a known bacterial vector using proceduressuch as described in T. Taniguchi et al., Proc. Natl Acad. Sci. USA,77:5230-5233 (1980). This exemplary bacterial vector could then betransformed into bacterial host cells and a BMP-17 and BMP-18-relatedprotein expressed thereby. For a strategy for producing extracellularexpression of BMP-17 or BMP-18-related proteins in bacterial cells, see,e.g. European patent application EPA 177,343.

Similar manipulations can be performed for the construction of an insectvector [See, e.g. procedures described in published European patentapplication 155,476] for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. [See, e.g., procedures described in publishedPCT application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a BMP-17 or BMP-18 or relatedprotein of the invention in mammalian cells may involve the constructionof cells containing multiple copies of the heterologous BMP-17 or BMP-18gene. The heterologous gene is linked to an amplifiable marker, e.g. thedihydrofolate reductase (DHFR) gene for which cells containing increasedgene copies can be selected for propagation in increasing concentrationsof methotrexate (MTX) according to the procedures of Kaufman and Sharp,J. Mol. Biol., 159:601-629 (1982). This approach can be employed with anumber of different cell types.

For example, a plasmid containing a DNA sequence for a BMP-17 or BMP-18or related protein of the invention in operative association with otherplasmid sequences enabling expression thereof and the DHFR expressionplasmid pAdA26SV(A)3 [Kaufman and Sharp, Mol. Cell. Biol., 2:1304(1982)] can be co-introduced into DHFR-deficient CHO cells, DUKX-BII, byvarious methods including calcium phosphate coprecipitation andtransfection, electroporation or protoplast fusion. DHFR expressingtransformants are selected for growth in alpha media with dialyzed fetalcalf serum, and subsequently selected for amplification by growth inincreasing concentrations of MTX (e.g. sequential steps in 0.02, 0.2,1.0 and 5 uM MTX) as described in Kaufman et al., Mol Cell Biol., 5:1650(1983). Transformants are cloned, and biologically active BMP-17 orBMP-18 expression is monitored by the Rosen-modified Sampath-Reddi ratbone formation assay described above in Example 3. BMP-17 and BMP-18protein expression should increase with increasing levels of MTXresistance. BMP-17 and BMP-18 polypeptides are characterized usingstandard techniques known in the art such as pulse labeling with [35S]methionine or cysteine and polyacrylamide gel electrophoresis. Similarprocedures can be followed to produce other related BMP-17 and BMP-18 orrelated proteins.

Example 5 Biological Activity of Expressed BMP-17 and BMP-18

To measure the biological activity of the expressed BMP-17 and BMP-18 orrelated proteins obtained in Example 4 above, the proteins are recoveredfrom the cell culture and purified by isolating the BMP-17, BMP-18 orrelated proteins from other proteinaceous materials with which they areco-produced as well as from other contaminants. The purified protein maybe assayed in accordance with the rat bone formation assay described inExample 3.

Purification is carried out using standard techniques known to thoseskilled in the art.

Protein analysis is conducted using standard techniques such as SDS-PAGEacrylamide [Laemmli, Nature 227:680 (1970)] stained with silver [Oakley,et al. Anal. Biochem. 105:361 (1980)] and by immunoblot [Towbin, et al.Proc. Natl. Acad. Sci. USA 76:4350 (1979)]

Example 6

Using Northern analysis, BMP-17, BMP-18 and related proteins can betested for their effects on various cell lines. Suitable cell linesinclude cell lines derived from E13 mouse limb buds. After 10 days oftreatment with BMP-17, BMP-18 or related protein, the cell phenotype isexamined histologically for indications of tissue differentiation. Inaddition, Northern analysis of mRNA from BMP-17, BMP-18 or relatedprotein treated cells can be performed for various markers including oneor more of the following markers for bone, cartilage and/ortendon/ligament, as described in Table IV:

TABLE IV Marker Bone Cartilage Tendon/Ligament Osteocalcin + − −Alkaline Phosphatase + − − Proteoglycan Core Protein +/−¹ + +² CollagenType I + + + Collagen Type II +/−¹ + +² Decorin + + + Elastin +/−³ ? +¹Marker seen early, marker not seen as mature bone tissue forms ²Markerdepends upon site of tendon; strongest at bone interface ³Marker seen atlow levels

Example 7 Embryonic Stem Cell Assay

In order to assay the effects of the BMP-17 and BMP-18 proteins of thepresent invention, it is possible to assay the growth anddifferentiation effects in vitro on a number of available embryonic stemcell lines. One such cell line is ES-El4TG2, which is available from theAmerican Type Culture Collection in Manassas, Va.

In order to conduct the assay, cells may be propagated in the presenceof 100 units of LIF to keep them in an undifferentiated state. Assaysare setup by first removing the LIF and aggregating the cells insuspension, in what is known as embryoid bodies. After 3 days theembryoid bodies are plated on gelatin coated plates (12 well plates forpCR analysis, 24 well plates for immunocytochemistry) and treated withthe proteins to be assayed. Cells are supplied with nutrients andtreated with the protein factor every 2-3 days. Cells may be adapted sothat assays may be conducted in media supplemented with 15% Fetal BovineSerum (FBS) or with CDM defined media containing much lower amounts ofFBS.

At the end of the treatment period (ranging from 7-21 days) RNA isharvested from the cells and analyzed by quantitative multiplex PCR forthe following markers: Brachyury, mesodermal marker, Ap-2, an ectodermalmarker, and HNF-3α a an endodennal marker. Through immunocytochemistry,it is also possible to detect the differentiation of neuronal cells(glia and neurons), muscle cells (cardiomyocytes, skeletal and smoothmuscle), and various other phenotype markers such as proteoglycan coreprotein (cartilage), and cytokeratins (epidermis). Since these cellshave a tendency to differentiate autonomously when LIF is removed, theresults are always quantitated by comparison to an untreated control.

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

7 1101 base pairs nucleic acid single linear cDNA pro_peptide 1..426mat_peptide 427..1098 CDS 1..1098 1 ATG CAG CCC CTG TGG CTC TGC TGG GCACTC TGG GTG TTG CCC CTG GCC 48 Met Gln Pro Leu Trp Leu Cys Trp Ala LeuTrp Val Leu Pro Leu Ala -142 -140 -135 -130 AGC CCC GGG GCC GCC CTG ACCGGG GAG CAG CTC CTG GGC AGC CTG CTG 96 Ser Pro Gly Ala Ala Leu Thr GlyGlu Gln Leu Leu Gly Ser Leu Leu -125 -120 -115 CGG CAG CTG CAG CTC AAAGAG GTG CCC ACC CTG GAC AGG GCC GAC ATG 144 Arg Gln Leu Gln Leu Lys GluVal Pro Thr Leu Asp Arg Ala Asp Met -110 -105 -100 -95 GAG GAG CTG GTCATC CCC ACC CAC GTG AGG GCC CAG TAC GTG GCC CTG 192 Glu Glu Leu Val IlePro Thr His Val Arg Ala Gln Tyr Val Ala Leu -90 -85 -80 CTG CAG CGC AGCCAC GGG GAC CGC TCC CGC GGA AAG AGG TTC AGC CAG 240 Leu Gln Arg Ser HisGly Asp Arg Ser Arg Gly Lys Arg Phe Ser Gln -75 -70 -65 AGC TTC CGA GAGGTG GCC GGC AGG TTC CTG GCG TTG GAG GCC AGC ACA 288 Ser Phe Arg Glu ValAla Gly Arg Phe Leu Ala Leu Glu Ala Ser Thr -60 -55 -50 CAC CTG CTG GTGTTC GGC ATG GAG CAG CGG CTG CCG CCC AAC AGC GAG 336 His Leu Leu Val PheGly Met Glu Gln Arg Leu Pro Pro Asn Ser Glu -45 -40 -35 CTG GTG CAG GCCGTG CTG CGG CTC TTC CAG GAG CCG GTC CCC AAG GCC 384 Leu Val Gln Ala ValLeu Arg Leu Phe Gln Glu Pro Val Pro Lys Ala -30 -25 -20 -15 GCG CTG CACAGG CAC GGG CGG CTG TCC CCG CGC AGC GCC CGG GCC CGG 432 Ala Leu His ArgHis Gly Arg Leu Ser Pro Arg Ser Ala Arg Ala Arg -10 -5 1 GTG ACC GTC GAGTGG CTG CGC GTC CGC GAC GAC GGC TCC AAC CGC ACC 480 Val Thr Val Glu TrpLeu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr 5 10 15 TCC CTC ATC GAC TCCAGG CTG GTG TCC GTC CAC GAG AGC GGC TGG AAG 528 Ser Leu Ile Asp Ser ArgLeu Val Ser Val His Glu Ser Gly Trp Lys 20 25 30 GCC TTC GAC GTG ACC GAGGCC GTG AAC TTC TGG CAG CAG CTG AGC CGG 576 Ala Phe Asp Val Thr Glu AlaVal Asn Phe Trp Gln Gln Leu Ser Arg 35 40 45 50 CCC CGG CAG CCG CTG CTGCTA CAG GTG TCG GTG CAG AGG GAG CAT CTG 624 Pro Arg Gln Pro Leu Leu LeuGln Val Ser Val Gln Arg Glu His Leu 55 60 65 GGC CCG CTG GCG TCC GGC GCCCAC AAG CTG GTC CGC TTT GCC TCG CAG 672 Gly Pro Leu Ala Ser Gly Ala HisLys Leu Val Arg Phe Ala Ser Gln 70 75 80 GGG GCG CCA GCC GGG CTT GGG GAGCCC CAG CTG GAG CTG CAC ACC CTG 720 Gly Ala Pro Ala Gly Leu Gly Glu ProGln Leu Glu Leu His Thr Leu 85 90 95 GAC CTT GGG GAC TAT GGA GCT CAG GGCGAC TGT GAC CCT GAA GCA CCA 768 Asp Leu Gly Asp Tyr Gly Ala Gln Gly AspCys Asp Pro Glu Ala Pro 100 105 110 ATG ACC GAG GGC ACC CGC TGC TGC CGCCAG GAG ATG TAC ATT GAC CTG 816 Met Thr Glu Gly Thr Arg Cys Cys Arg GlnGlu Met Tyr Ile Asp Leu 115 120 125 130 CAG GGG ATG AAG TGG GCC GAG AACTGG GTG CTG GAG CCC CCG GGC TTC 864 Gln Gly Met Lys Trp Ala Glu Asn TrpVal Leu Glu Pro Pro Gly Phe 135 140 145 CTG GCT TAT GAG TGT GTG GGC ACCTGC CGG CAG CCC CCG GAG GCC CTG 912 Leu Ala Tyr Glu Cys Val Gly Thr CysArg Gln Pro Pro Glu Ala Leu 150 155 160 GCC TTC AAG TGG CCG TTT CTG GGGCCT CGA CAG TGC ATC GCC TCG GAG 960 Ala Phe Lys Trp Pro Phe Leu Gly ProArg Gln Cys Ile Ala Ser Glu 165 170 175 ACT GCC TCG CTG CCC ATG ATC GTCAGC ATC AAG GAG GGA GGC AGG ACC 1008 Thr Ala Ser Leu Pro Met Ile Val SerIle Lys Glu Gly Gly Arg Thr 180 185 190 AGG CCC CAG GTG GTC AGC CTG CCCAAC ATG AGG GTG CAG AAG TGC AGC 1056 Arg Pro Gln Val Val Ser Leu Pro AsnMet Arg Val Gln Lys Cys Ser 195 200 205 210 TGT GCC TCG GAT GGT GCG CTCGTG CCA AGG AGG CTC CAG CCA 1098 Cys Ala Ser Asp Gly Ala Leu Val Pro ArgArg Leu Gln Pro 215 220 TAG 1101 366 amino acids amino acid linearprotein 2 Met Gln Pro Leu Trp Leu Cys Trp Ala Leu Trp Val Leu Pro LeuAla -142 -140 -135 -130 Ser Pro Gly Ala Ala Leu Thr Gly Glu Gln Leu LeuGly Ser Leu Leu -125 -120 -115 Arg Gln Leu Gln Leu Lys Glu Val Pro ThrLeu Asp Arg Ala Asp Met -110 -105 -100 -95 Glu Glu Leu Val Ile Pro ThrHis Val Arg Ala Gln Tyr Val Ala Leu -90 -85 -80 Leu Gln Arg Ser His GlyAsp Arg Ser Arg Gly Lys Arg Phe Ser Gln -75 -70 -65 Ser Phe Arg Glu ValAla Gly Arg Phe Leu Ala Leu Glu Ala Ser Thr -60 -55 -50 His Leu Leu ValPhe Gly Met Glu Gln Arg Leu Pro Pro Asn Ser Glu -45 -40 -35 Leu Val GlnAla Val Leu Arg Leu Phe Gln Glu Pro Val Pro Lys Ala -30 -25 -20 -15 AlaLeu His Arg His Gly Arg Leu Ser Pro Arg Ser Ala Arg Ala Arg -10 -5 1 ValThr Val Glu Trp Leu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr 5 10 15 SerLeu Ile Asp Ser Arg Leu Val Ser Val His Glu Ser Gly Trp Lys 20 25 30 AlaPhe Asp Val Thr Glu Ala Val Asn Phe Trp Gln Gln Leu Ser Arg 35 40 45 50Pro Arg Gln Pro Leu Leu Leu Gln Val Ser Val Gln Arg Glu His Leu 55 60 65Gly Pro Leu Ala Ser Gly Ala His Lys Leu Val Arg Phe Ala Ser Gln 70 75 80Gly Ala Pro Ala Gly Leu Gly Glu Pro Gln Leu Glu Leu His Thr Leu 85 90 95Asp Leu Gly Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala Pro 100 105110 Met Thr Glu Gly Thr Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp Leu 115120 125 130 Gln Gly Met Lys Trp Ala Glu Asn Trp Val Leu Glu Pro Pro GlyPhe 135 140 145 Leu Ala Tyr Glu Cys Val Gly Thr Cys Arg Gln Pro Pro GluAla Leu 150 155 160 Ala Phe Lys Trp Pro Phe Leu Gly Pro Arg Gln Cys IleAla Ser Glu 165 170 175 Thr Ala Ser Leu Pro Met Ile Val Ser Ile Lys GluGly Gly Arg Thr 180 185 190 Arg Pro Gln Val Val Ser Leu Pro Asn Met ArgVal Gln Lys Cys Ser 195 200 205 210 Cys Ala Ser Asp Gly Ala Leu Val ProArg Arg Leu Gln Pro 215 220 1101 base pairs nucleic acid single linearDNA (genomic) pro_peptide 1..405 mat_peptide 406..1098 CDS 1..1098 3 ATGTGG CCC CTG TGG CTC TGC TGG GCA CTC TGG GTG CTG CCC CTG GCT 48 Met TrpPro Leu Trp Leu Cys Trp Ala Leu Trp Val Leu Pro Leu Ala -135 -130 -125-120 GGC CCC GGG GCG GCC CTG ACC GAG GAG CAG CTC CTG GGC AGC CTG CTG 96Gly Pro Gly Ala Ala Leu Thr Glu Glu Gln Leu Leu Gly Ser Leu Leu -115-110 -105 CGG CAG CTG CAG CTC AGC GAG GTG CCC GTA CTG GAC AGG GCC GACATG 144 Arg Gln Leu Gln Leu Ser Glu Val Pro Val Leu Asp Arg Ala Asp Met-100 -95 -90 GAG AAG CTG GTC ATC CCC GCC CAC GTG AGG GCC CAG TAT GTA GTCCTG 192 Glu Lys Leu Val Ile Pro Ala His Val Arg Ala Gln Tyr Val Val Leu-85 -80 -75 CTG CGG CGC AGC CAC GGG GAC CGC TCC CGC GGA AAG AGG TTC AGCCAG 240 Leu Arg Arg Ser His Gly Asp Arg Ser Arg Gly Lys Arg Phe Ser Gln-70 -65 -60 AGC TTC CGA GAG GTG GCC GGC AGG TTC CTG GCG TCG GAG GCC AGCACA 288 Ser Phe Arg Glu Val Ala Gly Arg Phe Leu Ala Ser Glu Ala Ser Thr-55 -50 -45 -40 CAC CTG CTG GTG TTC GGC ATG GAG CAG CGG CTG CCG CCC AACAGC GAG 336 His Leu Leu Val Phe Gly Met Glu Gln Arg Leu Pro Pro Asn SerGlu -35 -30 -25 CTG GTG CAG GCC GTG CTG CGG CTC TTC CAG GAG CCG GTC CCCAAG GCC 384 Leu Val Gln Ala Val Leu Arg Leu Phe Gln Glu Pro Val Pro LysAla -20 -15 -10 GCG CTG CAC AGG CAC GGG CGG CTG TCC CCG CGC AGC GCC CAGGCC CGG 432 Ala Leu His Arg His Gly Arg Leu Ser Pro Arg Ser Ala Gln AlaArg -5 1 5 GTG ACC GTC GAG TGG CTG CGC GTC CGC GAC GAC GGC TCC AAC CGCACC 480 Val Thr Val Glu Trp Leu Arg Val Arg Asp Asp Gly Ser Asn Arg Thr10 15 20 25 TCC CTC ATC GAC TCC AGG CTG GTG TCC GTC CAC GAG AGC GGC TGGAAG 528 Ser Leu Ile Asp Ser Arg Leu Val Ser Val His Glu Ser Gly Trp Lys30 35 40 GCC TTC GAC GTG ACC GAG GCC GTG AAC TTC TGG CAG CAG CTG AGC CGG576 Ala Phe Asp Val Thr Glu Ala Val Asn Phe Trp Gln Gln Leu Ser Arg 4550 55 CCC CGG CAG CCG CTG CTG CTA CAG GTG TCG GTG CAG AGG GAG CAT CTG624 Pro Arg Gln Pro Leu Leu Leu Gln Val Ser Val Gln Arg Glu His Leu 6065 70 GGC CCG CTG GCG TCC GGC GCC CAC AAG CTG GTC CGC TTT GCC TCG CAG672 Gly Pro Leu Ala Ser Gly Ala His Lys Leu Val Arg Phe Ala Ser Gln 7580 85 GGG GCG CCA GCC GGG CTT GGG GAG CCC CAG CTG GAG CTG CAC ACC CTG720 Gly Ala Pro Ala Gly Leu Gly Glu Pro Gln Leu Glu Leu His Thr Leu 9095 100 105 GAC CTC AGG GAC TAT GGA GCT CAG GGC GAC TGT GAC CCT GAA GCACCA 768 Asp Leu Arg Asp Tyr Gly Ala Gln Gly Asp Cys Asp Pro Glu Ala Pro110 115 120 ATG ACC GAG GGC ACC CGC TGC TGC CGC CAG GAG ATG TAC ATT GACCTG 816 Met Thr Glu Gly Thr Arg Cys Cys Arg Gln Glu Met Tyr Ile Asp Leu125 130 135 CAG GGG ATG AAG TGG GCC AAG AAC TGG GTG CTG GAG CCC CCG GGCTTC 864 Gln Gly Met Lys Trp Ala Lys Asn Trp Val Leu Glu Pro Pro Gly Phe140 145 150 CTG GCT TAC GAG TGT GTG GGC ACC TGC CAG CAG CCC CCG GAG GCCCTG 912 Leu Ala Tyr Glu Cys Val Gly Thr Cys Gln Gln Pro Pro Glu Ala Leu155 160 165 GCC TTC AAT TGG CCA TTT CTG GGG CCG CGA CAG TGT ATC GCC TCGGAG 960 Ala Phe Asn Trp Pro Phe Leu Gly Pro Arg Gln Cys Ile Ala Ser Glu170 175 180 185 ACT GCC TCG CTG CCC ATG ATC GTC AGC ATC AAG GAG GGA GGCAGG ACC 1008 Thr Ala Ser Leu Pro Met Ile Val Ser Ile Lys Glu Gly Gly ArgThr 190 195 200 AGG CCC CAG GTG GTC AGC CTG CCC AAC ATG AGG GTG CAG AAGTGC AGC 1056 Arg Pro Gln Val Val Ser Leu Pro Asn Met Arg Val Gln Lys CysSer 205 210 215 TGT GCC TCG GAT GGG GCG CTC GTG CCA AGG AGG CTC CAG CCA1098 Cys Ala Ser Asp Gly Ala Leu Val Pro Arg Arg Leu Gln Pro 220 225 230TAG 1101 366 amino acids amino acid linear protein 4 Met Trp Pro Leu TrpLeu Cys Trp Ala Leu Trp Val Leu Pro Leu Ala -135 -130 -125 -120 Gly ProGly Ala Ala Leu Thr Glu Glu Gln Leu Leu Gly Ser Leu Leu -115 -110 -105Arg Gln Leu Gln Leu Ser Glu Val Pro Val Leu Asp Arg Ala Asp Met -100 -95-90 Glu Lys Leu Val Ile Pro Ala His Val Arg Ala Gln Tyr Val Val Leu -85-80 -75 Leu Arg Arg Ser His Gly Asp Arg Ser Arg Gly Lys Arg Phe Ser Gln-70 -65 -60 Ser Phe Arg Glu Val Ala Gly Arg Phe Leu Ala Ser Glu Ala SerThr -55 -50 -45 -40 His Leu Leu Val Phe Gly Met Glu Gln Arg Leu Pro ProAsn Ser Glu -35 -30 -25 Leu Val Gln Ala Val Leu Arg Leu Phe Gln Glu ProVal Pro Lys Ala -20 -15 -10 Ala Leu His Arg His Gly Arg Leu Ser Pro ArgSer Ala Gln Ala Arg -5 1 5 Val Thr Val Glu Trp Leu Arg Val Arg Asp AspGly Ser Asn Arg Thr 10 15 20 25 Ser Leu Ile Asp Ser Arg Leu Val Ser ValHis Glu Ser Gly Trp Lys 30 35 40 Ala Phe Asp Val Thr Glu Ala Val Asn PheTrp Gln Gln Leu Ser Arg 45 50 55 Pro Arg Gln Pro Leu Leu Leu Gln Val SerVal Gln Arg Glu His Leu 60 65 70 Gly Pro Leu Ala Ser Gly Ala His Lys LeuVal Arg Phe Ala Ser Gln 75 80 85 Gly Ala Pro Ala Gly Leu Gly Glu Pro GlnLeu Glu Leu His Thr Leu 90 95 100 105 Asp Leu Arg Asp Tyr Gly Ala GlnGly Asp Cys Asp Pro Glu Ala Pro 110 115 120 Met Thr Glu Gly Thr Arg CysCys Arg Gln Glu Met Tyr Ile Asp Leu 125 130 135 Gln Gly Met Lys Trp AlaLys Asn Trp Val Leu Glu Pro Pro Gly Phe 140 145 150 Leu Ala Tyr Glu CysVal Gly Thr Cys Gln Gln Pro Pro Glu Ala Leu 155 160 165 Ala Phe Asn TrpPro Phe Leu Gly Pro Arg Gln Cys Ile Ala Ser Glu 170 175 180 185 Thr AlaSer Leu Pro Met Ile Val Ser Ile Lys Glu Gly Gly Arg Thr 190 195 200 ArgPro Gln Val Val Ser Leu Pro Asn Met Arg Val Gln Lys Cys Ser 205 210 215Cys Ala Ser Asp Gly Ala Leu Val Pro Arg Arg Leu Gln Pro 220 225 230 15base pairs nucleic acid single linear DNA (genomic) 5 CATGGGCAGC TCGAG15 34 base pairs nucleic acid single linear DNA (genomic) 6 CTGCAGGCGAGCCTGAATTC CTCGAGCCAT CATG 34 68 base pairs nucleic acid single linearDNA (genomic) 7 CGAGGTTAAA AAACGTCTAG GCCCCCCGAA CCACGGGGAC GTGGTTTTCCTTTGAAAAAC 60 ACGATTGC 68

What is claimed is:
 1. A purified bone morphogenetic protein-17polypeptide comprising the amino acid sequence from amino acid #1 to#224 as set forth in SEQ ID NO:
 2. 2. A purified BMP-17 polypeptide ofclaim 1 wherein said polypeptide is a dimer and wherein at least onesubunit comprises the amino acid sequence from amino acid #1 to #224 ofSEQ ID NO:
 2. 3. A purified bone morphogenetic protein-17 polypeptideproduced by the steps of: (a)culturing a cell transformed with a DNAmolecule comprising a DNA sequence consisting of nucleotides #427 to#1098 of SEQ ID NO:1; and (b)recovering and purifying from said culturemedium a polypeptide comprising the amino acid sequence from amino acid#1 to amino acid #224 of SEQ ID NO:
 2. 4. A purified bone morphogeneticprotein-17 polypeptide according to claim 3, wherein said polypeptide isa dimer comprising two subunits, wherein one subunit comprises the aminoacid sequence from amino acid #1 to amino acid #224 of SEQ ID NO: 2, andone subunit comprises an amino acid sequence for a bone morphgeneticprotein selected from the group consisting of BMP-1, BMP-2, BMP-3,BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-15 and BMP-16.
 5. A purified bone morphgenetic protein-18polypeptide comprising the amino acid sequence from amino acid #1 to#231 as set forth in SEQ ID NO:
 4. 6. A purified BMP-18 polypeptide ofclaim 5 wherein said polypeptide is a dimer and wherein at least onesubunit comprises the amino acid sequence from amino acid #1 to #231 ofSEQ ID NO:
 4. 7. A purified bone morphogenetic protein-18 polypeptideproduced by the steps of: a) culturing a cell transformed with a DNAmolecule comprising a DNA sequence consisting of nucleotides #406 to#1098 of SEQ ID NO:3; and (b) recovering and purifying from said culturemedium a polypeptide comprising amino acid sequence from amino acid #1to amino acid #231 of SEQ ID NO:
 4. 8. A purified bone morphogeneticprotein-18 polypeptide according to claim 7, wherein said polypeptide isa dimer comprising two subunits, wherein one subunit comprises the aminoacid sequence from amino acid #1 to amino acid #231 of SEQ ID NO: 4, andone subunit comprises an amino acid sequence for a bone morphogeneticprotein selected from the group consisting of BMP-1, BMP-2, BMP-3,BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12,BMP-13, BMP-15 and BMP-16.
 9. Antibodies to a purified BMP-17 proteinaccording to claim 1, wherein the antibodies specifically bind to saidpurified BMP-17 protein.
 10. Antibodies to a purified BMP-18 proteinaccording to claim 5, wherein the antibodies specifically bind to saidpurified BMP-18 protein.
 11. A purified morphogenetic protein-17polypeptide comprising the amino acid sequence selected from the groupconsisting of amino acids #−142, −65, −7, 1, 109 or 124 to #211 or 224of SEQ ID NO:2.
 12. A purified bone morphogenetic protein-18 polypeptidecomprising the amino acid sequence selected from the group consisting ofamino acids #−135, −58, 1, 8, 116 or 131 to #218 or 231 of SEQ ID NO:4.